Since decades, tremendous efforts are made to collect pharmacologically active substances from natural organisms and to test their effects in various disease areas.
The sea hare Aplysia punctata produces a purple ink to protect itself from predation. This ink was shown to contain an anti-tumor activity (Butzke et al., 2004). Subsequently, studies were conducted to isolate the factor from crude ink, resulting in the discovery of APIT, the Aplysia Punctata Ink Toxin. Recently, the factor was cloned and characterized to be a weakly glycosylated FAD-binding L-amino acid oxidase that catalyzes the oxidative deamination of L-lysine and L-arginine and thereby produces hydrogen peroxide (H2O2), ammonium (NH4+) and the corresponding alpha-keto acids (Butzke et al., 2005).
L-amino acid oxidases (LAAOs, EC 1.4.3.2), can be found in secretions and venoms. Members of this family of flavoenzymes catalyze the stereospecific oxidative deamination of L-amino acids and thereby produce H2O2, ammonium and the corresponding alpha-keto acids (Du et al., 2002). The 3D individual LAAOs differ in their substrate specificity: Snake venom L-amino acid oxidases (sv-LAAOs) which constitute up to 30% (by weight) of the crude venom (Ponnudurai et al., 1994), possess a clear preference for hydrophobic amino acids. A fish capsule LAAO termed AIP (Apoptosis-Inducing Protein) which is induced by larval nematode infection of Scomber japonicus is highly specific for L-lysine (Jung et al., 2000). Achacin, a mucus LAAO from the African snail Achatina fulica, metabolizes a very broad range of substrates, including hydrophobic amino acids along with L-lysine, L-arginine, L-cysteine, L-asparagine and L-tyrosine (Ehara et al., 2002).
L-amino acid oxidases have not been investigated for their half-life after administration to mammalians at any routes. With respect to drug development, this is however an important issue and a prerequisite to efficiently exert a therapeutic effect. Data in this respect are only available for a D-amino acid oxidase employing D-proline as substrate (Fang et al., 2002). After intravenous injection, native D-amino acid oxidase was, however, rapidly cleared from the circulation. A pegylated derivative did not exhibit a significantly increased circulation half-life time which was well below 1 h. A product with such a short circulation half life time cannot be expected to have a substantial therapeutic benefit for therapy, e.g. for cancer therapy. Thus, according to the teaching of the prior art it would have been expected that pegylated amino acid oxidases would lack therapeutically efficacy.
An object of the present invention was to increase the therapeutic efficacy of L-amino acid oxidoreductases in order to provide a therapeutic product with improved characteristics.